适于黄瓜叶片蛋白质组分析的双向电泳方法最佳条件的研究

A Two-Dimensional Electrophoresis Protocol Suitable for Proteomic Analysis of Cucumber Leaves

为建立适于黄瓜叶片蛋白质组分析的双向电泳方法,对黄瓜叶片蛋白提取方法、裂解缓冲液、等电聚焦(IEF)参数进行研究。结果表明:丙酮法或三氯乙酸/丙酮法提取黄瓜蛋白质较好,2-DE图谱中蛋白点较多;裂解缓冲液为9.5mol·L-1尿素,2mol·L-1硫脲,1%二硫苏糖醇(DTT),2mmol·L-1三丁基磷(TBP),4%3-[3-(胆酰氨丙基)二甲铵基]丙磺酸内盐(CHAPS),0.2%两性电解质,0.002%溴酚蓝,0.25%吐温20,10%异丙醇,5%甘油,10mmol.L-1苯甲基磺酰氟(PMSF)时,电泳图谱效果最好;IEF等电聚焦条件为25000Vh时,2-DE图谱蛋白点多且清晰。

A 2-DE （tow-dimensional electrophoresis） protocol suited for the separation of proteins from cucumber leaves was established. Protein extraction, lysis buffer and IEF parameter were modified and improved to minimize markedly the interference by leaf pigments and other non-protein substances, thus resulting in satisfactory separation results. Cucumber leaves protein was extracted with DTT/ acetone or 20% w/v TCA in acetone, then resolubilized in lysis buffer （9.5mol·L^-1 urea, 2mol·L^-1 thiourea, 1% DTT, 2mmol·L^-1 TBP, 4% CHAPS, 0.2% Carrier ampholyte pH 3-10, 0.25% Tween 20, 10% Glycerol, 10mmol·L^-1 PMSF, 10mM PSMF）. Isoelectric focusing （IEF） was performed at 20℃ under the 25000Vh conditions.